Stable hydrophilic acridinium esters suitable for liposome encapsulation

ABSTRACT

A method for the detection of an analyte in a fluid sample using liposomes encapsulating acridinium esters (lumisomes). Hydrophilic polysubstituted aryl acridinium esters are useful as chemiluminescent markers and can be encapsulated within liposome vesicles without significant leakage of the esters from the vesicles. The lumisomes can be coupled to molecules with biological activity, such as antigens, antibodies, and nucleic acids, and used in luminescent assays.

This is a continuation of copending application Ser. No. 07/226,639filed on Aug. 1, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a novel method for the detection of ananalyte in a fluid sample. More particularly, the present inventionrelates to a method for the detection of an analyte in a fluid sampleusing acridinium esters encapsulated within the walls of liposomes(lumisomes). This invention also relates to novel acridinium estersuseful as chemiluminescent markers which can be encapsulated withinliposome vesicles without significant leakage of the esters from thevesicles.

BACKGROUND OF THE INVENTION

The use of liposomes as carriers of marker molecules for nonisotopicimmunoassays is known. See, e.g., U.S. Pat. Nos. 4,704,355; 4,695,554;4,656,129 and 4,193,983. An important advantage of using liposomes inimmunoassays is the ability of liposomes to carry a large number ofmarker molecules per liposome vesicle, and thereby provide an amplifiedsignal to immunoassays. Immunoassays utilizing liposomes withencapsulated macromolecular markers such as enzymes or small organicmarker molecules such as fluorescent or absorbing dyes, spin-labels,metal chelators, and enzyme activators or inhibitors, have beendescribed. See, e.g., Cricka, L. and Carter T., Clinical and BiochemicalLuminescence, pp. 153-178 (Marcel Dekker, Inc., New York and Basel,1982).

Prior to the present invention, chemiluminescent markers such as theacridinium esters described in Ann. Clin. Biochem. 25, p. 27 (1988),Clin. Chem. 31, p. 664 (1985), European Patent Application No. EP82,636, and U.S. Pat. No. 4,745,181, have only been used as labels forimmunoassays by conjugating them directly to biological molecules, suchas antigens or antibodies. The lipophilic nature of the prior artacridinium esters and other chemiluminescent compounds render themunsuitable for encapsulation within liposomes because of their rapidleakage through the liposome wall. Additionally, the limited watersolubility of prior art acridinium esters and other chemiluminescentcompounds only allow the encapsulation of a few marker molecules perliposome vesicle, resulting in relatively low signal amplification.

Accordingly, it is the purpose of the present invention to provide anovel method for detecting an analyte using acridinium esters. It isalso a purpose of the present invention to provide novel hydrophilicacridinium esters useful for encapsulation within liposomes for use aschemiluminescent markers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a standard curve for a Total T₄ Assay using lumisomes of thisinvention.

FIG. 2 is a standard curve for a Free T₄ Assay using lumisomes of thisinvention

FIG. 3 is a standard curve for a CKMB Assay using lumisomes of thisinvention.

FIG. 4 is a comparison of two standard curves for a TSH Assay, the topcurve respresenting a TSH assay using lumisomes of this invention andthe bottom curve representing a TSH assay using antibody directlylabeled with acridinium ester.

FIG. 5 is a comparison of two standard curves for a TSH Assay, the topcurve representing a TSH assay using lumisomes of this invention andshortened incubation times and the bottom curve representing a TSH assayusing antibody directly labelled with acridinium ester and normalincubation times.

DESCRIPTION OF THE INVENTION

The following terms as used in the specification and claims shall havethe following meanings:

Analyte--the compound or composition to be measured which can be aligand that is mono- or polyepitopic, antigenic, or haptenic. Theanalyte can be a piece of DNA or RNA.

Antigen--any substance capable of provoking an immune response invertebrates, particularly with the production of specific antibodies.

Hapten--an incomplete antigen, incapable by itself to provoke an immuneresponse but when suitably attached to another molecule becomes capableof producing antibodies which will specifically recognize the hapten.

Epitope--a specific chemical and spatial configuration which isspecifically recognized by an antibody.

Ligand--any compound for which a receptor naturally exists or can beprepared.

Ligand analog--a modified ligand which can compete with the analogousligand for a receptor, the modification providing means to join amodified ligand to another molecule.

Receptor--any compound capable of recognizing a particular spatial andpolar organization of a molecule, i.e., epitopic site. Illustrativereceptors include antibodies, enzymes, antibody fragments, such as Fabfragments, DNA or RNA fragments, lectins, complement components,conglutin, rheumatoid factors, hormones, avidin, staphylococcal proteinA, and the like.

Antiligand--a receptor for a ligand.

DNA probe--piece of DNA that recognizes specific DNA or RNA sequences byhydridizing to complementary DNA or RNA.

RNA probe--piece of RNA that recognizes specific DNA or RNA sequences byhydridizing to complementary DNA or RNA.

Liposomes--single or multicompartmented bodies obtained when lipids,particularily lipid mixtures, are dispersed in aqueous suspension. Thewalls or membranes are composed of a continuous lipid bilayer.

Lumisomes--liposomes comprising an encapsulated acridinium ester.

The acridinium esters useful in the method of the present invention canbe any acridinium ester which can be encapsulated within a liposome andwhich can generate a chemiluminescent signal. Preferred acridiniumesters include acridinium esters of the following formula: ##STR1##wherein R₁ is alkyl, alkenyl, alkynyl, aryl, or aralkyl, containing from0 to 20 heteroatoms, preferably nitrogen, oxygen, phosphorous or sulfur;

R₂, R₃, R₅, and R₇ are hydrogen, amino, alkoxyl, hydroxyl, --COOH,halide, nitro, ##STR2## wherein R is alkyl, alkenyl, alkynyl, aryl, oraralkyl, containing from 0-20 heteratoms;

R₄ and R₈ are hydrogen, alkyl, alkenyl, alkynyl, aralkyl, or alkoxyl;

X is an anion, preferably CH₃ SO₄ ⁻, OSO₂ F⁻, halide, ##STR3## wherein Ris as defined above; Q is ##STR4## I is an ionizable group; and n is atleast 1.

Preferably

R₁ is alkyl, alkenyl, alkynyl, aryl or aralkyl of from 1 to 24 carbonatoms;

R₂, R₃, R₅ and R₇ are hydrogen, amino, --COOH, cyano, hydroxyl, alkoxylof from 1 to 4 carbon atoms, nitro, halide, --SO₃, or --SCN;

R₄ and R₈ are preferably hydrogen or alkyl, alkenyl, alkynyl, oralkoxyl, of from 1 to 8 carbon atoms; X is halide; R₆ is--Q--R--I.sub.(n) ; Q is ##STR5## and R is alkyl, alkenyl, alkynyl,aryl, or aralkyl, of from 1 to 24 carbon atoms, containing from 0 to 20heteroatoms selected from the group consisting of nitrogen, oxygen,phosphorous, and sulfur.

An ionizable group for the purposes of this invention is any functionalgroup which retains a net positive or negative charge within a specificpH range. Preferably the functional group will retain a net positive ornegative charge within the range of pH 2-10 and, more preferably, withinthe range of pH 5-9. I can be any ionizable group provided that theionizable group is not deleterious to the encapsulation of theacridinium ester of this invention within the liposome. I is preferably--SO₃ H, --OSO₃ H, --PO(OH)₂, --OPO(OH)₂, and n is preferably about 1 toabout 20 and, preferably, less than about 10.

More preferably, R₁ is alkyl of from 1 to 10 carbon atoms; R₂, R₃, R₅,and R₇ are hydrogen, nitro, --CN, halide, alkoxyl of from 1 to 4 carbonatoms, amino, or --SO₃ H; and R₄ and R₈ are alkyl of from 1 to 4 carbonatoms.

Most preferably, R₁, R₄, and R₈ are methyl; R₂, R₃, R₅, and R₇ arehydrogen; X is bromide; R₆ is selected from the group consisting ofaminomethanesulfonic acid, 7-amino-1,3-naphthalenedisulfonic acid,S-(3-sulfopropyl)cysteine, 2-aminoethyl hydrogen sulfate,2-aminoethylphosphonic acid, and 2-aminoethyl dihydrogen phosphate.

The R₅ and R₆ position can be interchanged in the acridinium esters ofthis invention. Accordingly, the preferred acridinium esters of thisinvention include acridinium esters of the following formula: ##STR6##wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and X are as defined above.

The novel acridinium esters of the present invention are highly solublein water and can be encapsulated in liposomes at high concentrations.Once inside liposomes, the novel acridinium esters remain encapsulatedfor extended periods of time and do not leak appreciably.

It will be appreciated that although the novel acridinium esters of thepresent invention have been described with respect to their usefulnesswith liposomes, the novel acridinium esters of the present invention arealso useful in other applications where acridinium esters are utilized,such as labeling ligands or analytes (such as antigens); labeling thespecific binding partners of ligands or analytes (such as thecorresponding antibodies); or labelling nucleic acids and moleculescomprising nucleic acids.

Lumisomes useful in this invention can be prepared by any of the variousknown methods for producing either unilamellar liposome vesicles ormultilamellar liposome vesicles. Lumisomes are single ormulticompartmented bodies obtained when lipids or lipid mixtures aredispersed in an aqueous suspension containing the acridinium estersuseful in this invention.

As an example of one method for producing lumisomes, lipids aredissolved in a suitable organic solvent, such as chloroform, and placedinto a suitable vessel. A dry film of lipids is formed on the interiorsurface of the vessel by evaporation of the organic solvent. The aqueoussolution containing the acridinium esters to be entrapped within thelumisomes is then placed in the vessel in contact with the lipid film.The lipid film is then dispersed in the aqueous solution by vigorousagitation or sonication.

Numerous other methods exist for forming liposomes which are useful forproducing the lumisomes useful in this invention and it is left to theartisan to choose the method best suited for a desired use. A preferredmethod for manufacturing liposomes is disclosed in copending U.S.application Ser. No. 940,519, filed on Dec. 10, 1986, now U.S. Pat. No.4,933,121, incorporated by reference.

The lumisome can be derivatized with a ligand, ligand analog, oranti-ligand using known procedures in the art. Depending on the intendeduse of the lumisome, the ligand, ligand analog, or anti-ligand can be anantigen, hapten, antibody, nucleic acid, DNA, RNA, avidin or otherreceptor.

The lumisomes so formed can be used as tracers in assays in which ananalyte in a sample fluid is to be detected. The type of assay utilizedand/or the analyte to be detected will determine the ligand, ligandanalog or anti-ligand used to form the lumisome. For example, if acompetitive assay is used for determining an antigen or hapten, theligand or ligand analog employed will be either the analyte or itsanalog.

If a sandwich assay is to be used the ligand, ligand analog oranti-ligand employed would be specific for the analyte to be assayed.For example, an antibody, such as a monoclonal antibody, elicited inresponse to the analyte to be assayed, could be used to derivatize thelumisome.

An example of an assay for detecting a DNA or RNA probe using thelumisomes of this invention is as follows: The DNA or RNA probe istagged with a ligand such as a hapten or a biotinylated modifiednucleotide. The DNA or RNA probe is allowed to hybridize withcomplementary DNA or RNA and immobilized on a solid support. Theimmobilized probe is then reacted with lumisomes comprising a receptorfor the ligand, such as an antibody or if the probe is biotinylated,avidin. The lumisomes are ruptured and the amount of signal generated bythe encapsulated acridinium ester is measured.

Alternatively, a receptor can be utilized which recognizes and binds tothe probe/complementary DNA or RNA hybrid, in the absence of a tag, suchas an anti-hybrid antibody.

The following Examples are presented to illustrate the presentinvention.

EXAMPLE 1 Preparation of 2',6'-Dimethyl-4'-carboxyphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH)

A mixture of 2',6'-dimethyl-4'-benzyloxycarbonylphenyl10-methyl-acridinium-9-carboxylate methosulfate (7.9, 13.4 mmole)(prepared as described in U.S. Pat. No. 4,745,181), 150 ml of glacialacetic acid and 46 ml of 48% hydrogen bromide was heated at 100° C.-105°C. for 3 hours and then left at room temperature overnight. 400 ml ofanhydrous ethyl ether was added to the mixture to form a second mixturewhich was then refrigerated 3 days. The second mixture was then filteredto produce a yellow crystalline residue. The residue was washed withanhydrous ethyl ether and air dried.

EXAMPLE 2 Preparation of 2',6'-Dimethyl-4'-(sulfomethylcarbamoyl)phenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-AMS)

A solution of 2'6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 20 mg, 0.043mmole) in 2 ml of dimethylformamide (DMF)/CHCl₃ (1:1) was cooled in anice bath, treated with triethylamine (31 ul, 0.215 mmole) and ethylchloroformate (6.1 ul, 0.065 mmole) to form a reaction mixture. After 30min. the reaction mixture was evaporated. The residue of the evaporationwas reconstituted in 2 ml of DMF, treated with triethylamine (31 ul,0.215 mmole) and aminomethanesulfonic acid (9.5 mg, 0.086 mmole) to forma second reaction mixture. The second reaction mixture was stirred atroom temperature overnight, and evaporated. The crude product so formedwas purified on one analytical TLC plate (Silica gel 60, F254, Merck &Co., Inc., Rahway, N.J.), and developed with chloroform/methanol/water(65:25:4). The yellow band which developed on the plate (Rf=0.38) (whichcould also be detected under both long and short UV light) was strippedfrom the plate and eluted with the same solvent system. The eluent soproduced was evaporated and the residue of the evaporation trituratedwith methanol to form a mixture. This mixture was then filtered througha polycarbonate membrane (13 mm diameter, 0.2 um pore size) (NucleoporeCorp., Pleasanton, Calif.) mounted on a syringe filter holder, and thefiltrate so produced was evaporated to give DMAE-AMS (15 mg, 62%).

EXAMPLE 3 Preparation of2',6'-Dimethyl-4'-[N-7-(1,3-disulfonaphthalenyl)carbamoyl]phenyl10-methyl-acridinium-9-carboxylate Bromide (DMAE-ANDS)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 18 mg, 0.038mmole) in 3.6 ml of a dioxane/water (1:1) mixture was treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (37 mg,0.193 mmole, Aldrich Chemical Co., Inc., Milwaukee, Wis.) at roomtemperature for 5 minutes to form a reaction mixture. A solution of7-amino-1,3-naphthalenedisulfonic acid (ANDS, 26 mg, 0.076 mmole,Aldrich Chemical Co., Inc.) in 0.9 ml of water was added to the reactionmixture which was then stirred at room temperature overnight andevaporated.

The residue of the evaporation was dissolved in a minimal amount of 0.1Msodium carbonate to obtain a solution at neutral pH. This solution wasmixed with an equal amount of methanol and purified on a 20×20 cmpreparative TLC plate (Silica gel 60, F254, Merck & Co., Inc.), anddeveloped with chloroform/methanol/water (55:40:5). The yellow bandwhich developed (Rf=0.4) was treated in the same manner as the yellowband described in Example 1 to give DMAE-ANDS (15 mg, 50%). Fast AtomBombardment (FAB) Mass Spectral Analysis (by Oneida Research Services,Whitesboro, N.Y.) in the positive ion mode gave a M+ peak of 671, a M+Napeak of 693, and a M+2Na peak of 715. A bromide peak of 80 was detectedin the negative ion mode.

EXAMPLE 4 Preparation of2',6'-Dimethyl-4'-{N-[1-carboxyl-2-(3-sulfopropylthio)ethyl]carbamoyl}phenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-SCYS)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 50 mg, 0.107mmole) in 10 ml of DMF/CHCl₃ (1:1) was cooled in an ice bath, treatedwith triethylamine (77 u., 0.535 mmole) and ethyl chloroformate (20 ul,0.214 mmole) to form a reaction mixture. After 30 min. the reactionmixture was evaporated. The residue of the evaporation was reconstitutedin 10 ml of DMF/CHCl₃ (1:1), treated with triethylamine (77 u., 0.535mmole), and S-3-sulfopropyl-L-cysteine (51 mg, 0.214 mmole) (prepared bythe method of U. T. Ruegg and J. Rudinger, J. Peptide Protein Res. 6,447, 1974) to form a second reaction mixture. The second reactionmixture was heated at 60° C.-70° C. overnight, and evaporated.

The residue of the evaporation was purified on one 20×20 cm preparativeTLC plate (Silica gel 60, F254, Merck & Co., Inc.), developed withchloroform/methanol/water (55:40:5). The yellow band which developed(Rf=0.4) (which could also be detected under both long and short UVlight) was treated in the same manner as the yellow band described inExample 1 to give the DMAE-SCys (37 mg, 36%).

FAB Mass Spectral Analysis in the positive ion mode gave a M+ peak of611, a M+Na peak of 633, and a M+Na,K peak of 673.

EXAMPLE 5 Preparation of2',6'-Dimethyl-4'-[N-(2-sulfonyloxyethyl)carbamoyl]phenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-AEOS)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 35 mg, 0.075mmole) in 5 ml of DMF was cooled in an ice bath, treated withtriethylamine (44 ul, 0.31 mmole) and ethyl chloroformate (8.5 ul, 0.090mmole). After 20 minutes, aminoethyl hydrogen sulfate (Aldrich, 30 mg,0.21 mmole) was added to form a reaction mixture, and the ice bathremoved. The reaction mixture was stirred at room temperature overnightand evaporated. The residue of the evaporation was triturated with 5 mlof a chloroform/methanol/water (73:24:3) mixture, and filtered to removethe insoluble materials. The filtrate so produced was concentrated,purified on a 20×20 cm preparative TLC plate (Silica gel 60, F254, Merck& Co., Inc.) and developed with chloroform/methanol/water (65:25:4). Theyellow band which developed (Rf=0.48) (which could also be detectedunder both long and short UV light) was treated in the same manner asthe yellow band described in Example 1 to give DMAE-AEOS (14 mg, 32%).

EXAMPLE 6 Preparation of2',6'-Dimethyl-4'-[N-(2-phosphonoethyl)carbamoyl]phenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-AEP)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 100 mg, 0.215mmole) in 20 ml of 25% DMF in chloroform was cooled in an ice bath,treated with triethylamine (180 ul, 1.30 mmole) and ethyl chloroformate(62 ul, 0.65 mmole) to form a reaction mixture. After 30 minutes, thereaction mixture was evaporated. The residue of the evaporation wasreconstituted in 12 ml of DMF. To this solution so formed was addedtriethylamine (300 ul, 2.15 mmole) and 2-aminoethylphosphonic acid (80mg, 0.64 mmole, Aldrich Chemical Co., Inc.) in 5 ml of water to form asecond reaction mixture. The second reaction mixture was stirred at roomtemperature overnight and evaporated. The residue of the evaporation wastaken up in 2-3 ml of chloroform/methanol/water (73:24:3), purified onone 20×20 cm preparative TLC plate (Silica gel 60, F254, Merck & Co.,Inc.) and developed with chloroform/methanol/water (65:25:4). The yellowband which developed on the plate (Rf=0.45) (which can be detected alsounder long and short UV light) was treated in the same manner as theyellow band described in Example 1 to give DMAE-AEP (72 mg, 58%).

FAB Mass Spectral analysis in the positive ion mode gave a M+ peak of493 and a M+Na peak of 515.

EXAMPLE 7 Preparation of2',6'-Dimethyl-4'[N-(2-phosphonooxyethyl)carbamoyl]phenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-AEOP)

A solution of 2',6'-dimethyl-4'-carboxylphenyl10-methyl-acridinium-9-carboxylate bromide (DMAE-COOH, 100 mg., 0.215mmole) in 20 ml of 25% DMF in chloroform was cooled in an ice bath,treated with triethylamine (180 ul, 1.30 mmole) and ethyl chloroformate(62 ul, 0.65 mmole) to form a reaction mixture. After 30 minutes, thereaction mixture was evaporated. The residue of the evaporation wasreconstituted in 12 ml of DMF. To this solution so formed was addedtriethylamine (300 ul, 2.15 mmole) and 2-aminoethyl dihydrogen phosphate(91 mg, 0.64 mmole, Aldrich Chemical Co., Inc.) in 5 ml of water to forma second reaction mixture. The second reaction mixture was stirred atroom temperature overnight and evaporated. The residue of thisevaporation was taken up in 2-3 ml of chloroform/methanol/water(73:24:3), purified on one 20×20 cm preparative TLC plate (Silica gel60, F254, Merck & Co., Inc.) and developed withchloroform/methanol/water (65:25:4). The yellow band which developed (Rf=0.45) (which could also be detected also under long and short UV light)was treated in the same manner as the yellow band described in Example 1to give DMAE-AEOP. (100 mg, 79%).

FAB Mass Spectral analysis in the positive ion mode gave a M+ peak of509.

EXAMPLE 8 Rate of Leakage of Acridinium Esters From Lumisomes

Lumisomes were prepared as follows:

Chloroform solutions containing 25 mg dipalmitoyl phosphatidylcholine,13.5 mg cholesterol, and 2.2 mg dipalmitoyl phosphatidylglycerol wereair-dried on a round flat glass dish, 7 cm in diameter, and were placedfor 16 hours in a vacuum to produce dry lipid films. Acridinium estersDMAE-COOH, DMAE-AMS, and DMAE-ANDS, were each dissolved in a solution of0.215M sucrose, 0.25 mM EDTA, pH 7 (0.1-0.26 mg/ml). Acridinium estersDMAE-SCYS, DMAE-AEOS, DMAE-AEP, and DMAE-AEOP, were each dissolved in asolution of 50mM sodium phosphate, pH 7.4 (0.1-0.26 mg/ml). A three mlaliquot of each acridinium ester solution so formed was added to aseparate dry lipid film and gently mixed at 45° C. for 10 min. toproduce a lumisome suspension. Each suspension so formed containingDMAE-COOH, DMAE-AMS, and DMAE-ANDS, was extruded through polycarbonatemembranes with a pore size of 0.4 and 0.2 microns and then washed 4times by ultracentrifugations at 45,000 rpm for 30 min. in TRIS Buffer(0.1 M TRIS, 0.03M NaCl, 0.01% NaN₃, 0.5 mM EDTA, pH 7.8) to produce alumisome pellet. The lumisome pellets so formed were each resuspended in10 ml of TRIS Buffer. Each lumisome suspension containing DMAE-SCYS,DMAE-AEOS, DMAE-AEP, and DMAE-AEOP, was extruded through polycarbonatemembranes with a pore size of 0.4 and 0.2 microns and then washed 4times by ultracentrifugation at 45,000 rpm for 30 min. in 50 mM sodiumphosphate, pH 7.4, to produce a lumisome pellet. The lumisome pellets soformed were each resuspended in 10 ml of 50 mM sodium phosphate, pH 7.4.A sample of each resuspended lumisome pellet was mixed with 0.1% TRITONX-100 detergent (weight/volume) for determination of the total amount ofencapsulated acridinium ester. Aliquots of each of the resuspendedlumisome pellets were incubated for 7 days at 4° C. and 37° C. Thealiquots were then centrifuged at 45,000 rpm for 2 hours. Aftercentrifugation, a sample was taken from each supernatant and mixed with0.1% TRITON X-100. The concentration of acridinium ester in eachsupernatant was then measured using the Ciba Corning Magic® LITEAnalyzer (Ciba Corning Diagnostics Corp., Medfield, Mass.). The percentleakage of acridinium ester was then calculated by comparing the amountof acridinium ester in the supernatant to the total amount of acridiniumester originally encapsulated within the lumisomes.

                  TABLE 1                                                         ______________________________________                                        Leakage of Acridinium Esters From Lumisomes                                   Acridinium     % Leakage at                                                                             % Leakage at                                        Ester          4° C./7 Days                                                                      37° C./7 Days                                ______________________________________                                        DMAE-COOH      25.0%      36.0%                                               DMAE-AMS       0.8%       26.0%                                               DMAE-ANDS      1.3%       1.9%                                                DMAE-SCYS      0.5%       4.9%                                                DMAE-AEOS      1.4%       52.8%                                               DMAE-AEP       0.3%       1.0%                                                DMAE-AEOP      1.8%       1.7%                                                ______________________________________                                    

EXAMPLE 9 Effect of Different Buffers on Acridinium Ester Leakage FromLumisomes

Lumisomes encapsulating DMAE-ANDS were prepared according to theprocedure described in Example 8 except that the DMAE-ANDS was dissolvedin the four buffers listed in Table 2, prior to addition to the drylipid film. The buffers were then used to wash, resuspend and store,respectively, the four lumisome prepartions in Table 2. The lumisomes soprepared were stored in their respective buffers for 35 days at either4° C. or 37° C. and then centrifuged at 45,000 rpm for 2 hours. Theconcentration of DMAE-ANDS in each supernatant was then measured using aCiba Corning Magic® LITE Analyzer. The percent leakage of DMAE-ANDS wasthen calculated by comparing the amount of DMAE-ANDS in the supernatantversus the amount of DMAE-ANDS originally encapsulated within thelumisomes.

                  TABLE 2                                                         ______________________________________                                        Effect of Different Buffers on the Leakage of DMAE-ANDS                       from Lumisomes After 35 Days at 4° C. and at 37° C.             Buffer (50 mM)                                                                            % Leakage at 4° C.                                                                   % Leakage at 37° C.                          ______________________________________                                        Sodium phosphate                                                                           0.35%         0.85%                                              pH 7.4                                                                        Sodium acetate                                                                            0.33          1.39                                                pH 7.4                                                                        Sodium tartarate                                                                          0.62          1.30                                                pH 7.4                                                                        Sodium HEPES                                                                              0.79          1.71                                                pH 7.4                                                                        ______________________________________                                    

EXAMPLE 10 Total T₄ Assay

A. Reagent preparation

Thyroxine (T₄) lumisomes encapsulating DMAE-AMS were prepared asdescribed in Example 8 except that 0.16 mg ofdipalmitoyl-phosphatidylethanolamine-succinyl-thyroxine (prepared asdescribed in U.S. application Ser. No. 094,667, filed on Sep. 9, 1987,herein incorporated by reference) was added to the lipid mixture.DMAE-AMS at 0.2 mg/ml in phosphate buffer was used for hydration of thelipid film. The final lumisome preparation was diluted in Buffer A(0.02M Tris, 0.1M NaCl, 0.001M EDTA, 0.1% bovine serum albumin (BSA),0.1% sodium azide, pH 7.8).

Monoclonal anti-T₄ antibody was produced in mice (A/J) by immunizationwith a BSA-T₄ conjugate and subsequent fusion of the splenocytes withSp2/0-Ag 14 myeloma cells by the procedure described by Kohler andMilstein in Nature (London), Vol. 256, pp. 495-497 (1975). Hybridomacells secreting anti-T₄ antibody were detected by radioimmunoassay usingthe following procedure: Supernatant from the cells were diluted 1:5 inphosphate buffered saline containing 0.1% (weight/volume) bovine gammaglobulin. 100 ul of each diluted supernatant and 100 ul of ¹²⁵I-labelled T₄ were added to a test tube and were incubated for one (1)hour at room temperature. Goat anti-mouse IgG coupled to paramagneticparticles were added to each tube for ten (10) minutes at roomtemperature. The particles were magnetically separated and counted. Thecells that tested positive (i.e., produced counts over background) wereplated at 0.1 cell/well and then retested after growth.

Cells resulting from this regrowth which tested positive were theninjected introperitoneally into pristane-primed mice (CAF). Asciticfluid from these mice was collected after 3-5 weeks. The anti-T₄antibody was purified from the ascitic fluid by Protein A columnchromatography using the Affi-Gel Protein A MAPS II Kit (Bio-RadLaboratories, Richmond, Calif.) according to the written protocolprovided by the kit. The anti-T₄ antibody was immobilized onparamagnetic particles as described by Groman et al, BioTechniques3:156-160 (1985). The antibody-derivatized particles were diluted to aconcentration of 50 ug of particles per 0.1 ml of Buffer B (0.03Mphosphate, 0.1M NaCl, 2.9 mg/ml merthiolate, and 0.1% BSA, 0.1% sodiumazide, pH 7.4).

B. Assay procedure

A series of standards (0.05 ml) with known increasing amounts of T₄ wereadded to 12×75 mm plastic tubes. 0.1 ml of the T₄ -lumisomes prepared inA containing 10×10⁶ RLU (relative light units) in Buffer A were thenadded, followed by the addition of 0.5 ml of the paramagnetic-particlesimmobilized with monoclonal anti-T₄ antibody as prepared in A. The tubeswere incubated for 1 hour at room temperature and then placed in amagnetic field of a specially designed rack useful for magneticseparation of paramagnetic particles in test tubes (available from CibaCorning Diagnostics Corp., Medfield, Mass.). The magnetic fieldseparated the particles from the supernatant and the supernatant wasthen decanted. The particles were washed once in 1 ml ofphosphate-buffered saline, vortexed, and magnetically separated. Theparticles were resuspended in 0.1 ml of TRITON X-100, 0.1% (w/v) indeionized water.

The tubes were then placed in a luminometer (MAGIC® LITE Analyzer, CibaCorning Diagnostics Corp., Medfield, Mass.). 0.3 ml of a solution of0.1% hydrogen peroxide in 0.1N HNO₃ was added to each tube by theluminometer and the light emission was triggered by the addition of 0.3ml of 0.25N NaOH containing ARQUAD surfactant (Armark Chemicals,Chicago, Ill.). The measured RLU for each tube was plotted against itsrespective T₄ concentration as shown in FIG. 1.

EXAMPLE 11 Free T₄ Assay

A series of serum-based standards containing known increasingconcentrations of free T₄ were added to 12×75 mm plastic tubes. 0.1 mlof the T₄ -lumisomes prepared in Example 10A containing 10×10⁶ RLU wasadded, followed by the addition of 0.5 ml of the paramagnetic-particlesimmobilized anti-T₄ antibody as prepared in Example 10A, in Buffer Bminus merthiolate. The reaction was carried out for 1 hour at roomtemperature and the tubes were further processed and read as in Example10. FIG. 2 shows the standard curve obtained by plotting the measuredRLU for each tube against its respective free T₄ concentration.

EXAMPLE 12 Creatine kinase MB (CKMB) Assay

A. Reagent preparation

Monoclonal antibodies to creatine kinase MB (CKMB) and creatine kinaseBB (CKBB) were prepared as described by Piran et al, Clinical Chemistry33:1517-1520 (1987).

Lumisomes were prepared as described in Example 8, except that 1 mgdithiopyridyl dipalmitoyl phosphatidylethanolamine (DTP-DPPE), preparedaccording to Barbet et al, J. Supramolecular Structure 16:243-258(1981), was added to the lipid mixture. The dry lipid film was hydratedwith DMAE-ANDS, 2 mg/ml, and the final lumisome preparation was dilutedin 0.1M sodium phosphate, 0.15M NaCl, and 0.005M EDTA, pH 7.5.

The anti-CKMB monoclonal antibody was coupled to the DTP-DPPE containinglumisomes by the method of Barbet et al J. Supromolecular Structure16:243-258 (1981). The anti-CKMB antibody bearing lumisomes were dilutedin Buffer C (0.1M 1,4-piperazinediethanesulfonic acid, 0.15M NaCl,0.001M EDTA, 0.1% NaN₃, 0.1% BSA, pH 6.5).

The anti-CKBB monoclonal antibody was immobilized on paramagneticparticles as described in Example 10 for the anti-T₄ antibody anddiluted in Buffer C to a final concentration of 100 ug of particles perml of Buffer C.

B. Assay procedure

A series of serum-based standards (0.1 ml) with known increasingconcentrations of human cardiac CKMB were added to 12×75 mm plastictubes. 0.1 ml of the anti-CKMB antibody bearing lumisomes prepared in Acontaining 10×10⁶ RLU was added to each tube and the tubes were thenincubated for 30 minutes at room temperature. To each tube was thenadded 50 ug of the anti-CKBB antibody bearing paramagnetic-particlesprepared in A and the the tubes were incubated for another 30 minutes atroom temperature. The tubes were then processed and read as in Example10, except that Buffer C minus BSA was used in the wash step instead ofphosphate-buffered saline. The measured RLU for each tube was plottedagainst its respective CKMB concentration as shown in FIG. 3.

C. Four samples of human sera were assayed using the procedure describedin B and concentrations of <1, 21, 24 and 68 ng/ml CKMB respectively,were determined. When these four samples were assayed using the CKMBMagic® LITE Assay (Ciba Corning Diagnostics Corp., Medfield, Mass.02052), CKMB values of <1, 18, 24 and 72 ng/ml, respectively weredetermined, indicating good agreement between the two assays.

EXAMPLE 13 Thyroid Stimulating Hormone (TSH) Assay

A. Reagent preparation

Anti-TSH monoclonal antibodies were produced from TSH-immunized mice asdescribed in Example 10 for the preparation of anti-T₄ antibodies.Preparation of DTP-DPPE containing lumisomes loaded with DMAE-ANDS andparamagnetic particles-immobilized anti-TSH was done as described inExample 12. These reagents were diluted in Buffer C.

B. Assay procedure

A series of serum-based standards (0.1 ml) with known increasingconcentrations of TSH were added to 12×75 mm plastic tubes. 0.1 ml ofthe anti-TSH antibody bearing lumisomes prepared in A containing 8×10⁶RLU were added and the tubes were then incubated for 2 hours at roomtemperature. Paramagnetic-particles immobilized anti-TSH antibody asprepared in A was then added (0.5 ml) and the tubes were then incubatedfor an additional 30 minutes at room temperature. The tubes were thenprocessed and read as described in Example 11.

C. The TSH assay was performed by the same procedure as described in B,except that the anti-TSH antibody bearing lumisomes were replaced by theanti-TSH antibody prepared in A labeled directly with2',6'-dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl10-methyl-acridinium-9-carboxylate methosulfate (DMAE-NHS).

D. The standard curves obtained by the procedures described in B and Cusing the two types of labels are shown in FIG. 4. The results indicatethat by using lumisomes encapsulating the hydrophilic acridinium esteranalogue as the label, the signal to noise ratio at the low end of theassay is significantly increased. This increase in signal to noise ratiois advantageous because it allows better assay sensitivity, precisionand speed.

E. The assay described in B was conducted using shorter incubation times(see FIG. 5). It was found that even when the first incubation time was2.5 minutes and the second incubation time was also 2.5 minutes thesensitivity of the assay was satisfactory. It was not possible to obtaina sufficiently sensitive standard curve using the anti-TSH antibodywhich had been labeled directly with DMAE-NHS using the short incubationtimes. Surprisingly, the signal to noise ratio of the short lumisomeassay was higher than that of the 2.0 hour/0.5 hour conventional assay(FIG. 5).

What is claimed is:
 1. An acridinium ester of the formula: ##STR7##wherein R₁ is CH₂ A where C is a carbon atom and where A is hydrogen,alkyl, alkenyl, alkynyl, aryl or aralkyl, with R₁ having up to 24carbons and up to 20 heteroatoms selected from the group consisting ofnitrogen, oxygen, phosphorous and sulfur;R₂, R₃, R₅ and R₇ are hydrogen,amino, alkoxyl, hydroxyl, --COOH, halide, nitro, --CN, --SO₃ H, ##STR8##or --SCN, wherein R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, havingup to 24 carbons and up to 20 heteroatoms selected from the groupconsisting of nitrogen, oxygen, phosphorous and sulfur; R₄ and R₈ arealkyl, alkenyl, alkynyl, aralkyl, or alkoxyl having up to 8 carbons,with no branching wherein the side-chain groups have more than 2carbons; X is an anion; R₆ is --R--I.sub.(n) or --Q--R--I.sub.(n),wherein R is defined as above; Q is ##STR9## --O--, --S--, --NH--, or--SO₃ --; I is selected from the group consisting of --SO₃ H, --OSO₃ H,--PO(OH)₂ or --OPO(OH)₂ ; and n is 1-4.
 2. An acridinium ester of claim1 wherein R₄ and R₈ are alkyl, alkenyl, alkynyl, aralkyl, or alkoxylhaving up to 8 carbons, with no branching in a given group if more than4 carbons are present in that group.
 3. An acridinium ester of claim 1wherein X is CH₃ SO₄ ⁻, OSO₂ F⁻, halide, OSO₂ CF₃ ⁻, OSO₂ C₄ F₉ ⁻ or##STR10##
 4. An acridinium ester of claim 1 whereinR₄ and R₈ are alkyl,alkenyl, alkynyl, aralkyl, or alkoxyl having up to 8 carbons, with nobranching in a given group if more than 4 carbons are present in thatgroup; and X is CH₃ SO₄ ⁻, OSO₂ F⁻, halide, OSO₂ CF₃ ⁻, OSO2C₄ F₉ ⁻ or##STR11##
 5. An acridinium ester of claim 1 wherein R₂, R₃, R₅ or R₇ aredefined in claim 1, except that R has up to 4 carbons and up to 5heteroatoms.
 6. An acridinium ester of claim 1 wherein the I groups inR₆ are different from each other.
 7. An acridinium ester of claim 1wherein the positions of R₅, R₆, and R₇ are interchanged.
 8. Anacridinium ester selected from the group consisting of DMAE-AMS,DMAE-ANDS, DMAE-SCYS, DMAE-AEOS, DMAE-AEP, or DMAE-AEOP.